It is vitally important to develop novel analytical tools and approaches for reliable identification and quantitation of protein posttranslational modifications (PTMs) such as phosphorylation. The existing approaches are mostly based on the combination of mass spectrometry with liquid chromatography (LC-MS). LC plays a role of a separation platform although its analytical capabilities can add an invaluable piece of information about chemical and physical properties of proteins. The purpose of this project is to develop a commercially viable analytical platform that will integrate two sequence related data streams from LC and tandem mass spectrometry combination. The main feature of the proposed platform, which will include a combination of PTM characterization LC-MS hardware and a set of developed software tools, is the utilization of the novel Liquid Chromatography of Biomacromolecules at Critical Conditions (BioLCCC) technology for sequence-dependent retention time prediction for peptides with PTMs. The new approach is expected to increase the throughput of modified protein analysis, the dynamic range of analyzed species, and to allow rapid, unambiguous identification of the sites of modifications along the protein amino acid sequences. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The application of proteomics tools plays an important role in modern basic science, drug discovery and clinical applications. We propose a new platform for phosphoprotein identification using liquid separation, refined enrichment technique, and multimode tandem mass spectrometry to increase reliability and throughput in proteomics. [unreadable] [unreadable] [unreadable]